Today binding-pair assays are utilized extensively in such fields as clinical, forensic, and veterinary medicine, pharmacological testing, environmental monitoring, food quality assurance, and other related areas. All these fields have needs for rapid and effective analysis of specific substances (referred to as analytes), which are frequently found in low concentrations within the given test sample. The basic principles and mechanisms of assays are specifically designed to accommodate such urgent needs.
These assays are especially useful in the in vitro determination of the presence and concentration of analytes within physiological fluids. For example, the determination of specific proteins, enzymes, hormones, metabolites, and therapeutic or toxic drugs within the blood, urine, or cerebral spinal fluid has greatly enhanced the efficacy of diagnostic methodologies in clinical medicine.
Moreover, the development of non-radioactive labeling components, which allow the direct visualization of the completed reaction, has facilitated the use of binding-pair assay procedures outside of the "typical" laboratory. For example, in clinical office settings, non-radioactively-labeled, binding-pair assays are useful for providing rapid, simple procedures which may be performed while the patient is still in the office. Thus diagnosis can be accomplished without delay, and treatment may be instituted during a single visit.
Without such assays, it was frequently necessary to collect the sample from the patient during a first visit and to have the biological sample analyzed by a clinical laboratory at a later time. During such time, the patient was sent home and often required to return for a second office visit in order to receive the proper treatment and/or medication. Such delay was at best inefficient, and at worst potentially life threatening.
The term "binding-pair assay" refers to an assay between two binding-pair members designed to facilitate the formation of a complex between a particular analyte of interest and another substance capable of specific interaction with the analyte. In this way, the presence of particular analyte may be detected. Alternatively, the binding-pair member may be a substance which, if detectable, may be used to infer the presence or absence of the analyte within the sample.
In the context of the present invention, the term "analyte" refers to, but is not limited to, compounds such as proteins, modified proteins, peptides, nucleic acids such as deoxyribonucleic acid (DNA), ribonucleic acid (RNA), peptide nucleic acid (PNA), haptens, antigens, antibodies, and any metabolites of these substances and any other compounds, either natural or synthetic, which may be of diagnostic interest and which have a specific binding partner (e.g., the receptor moiety of a ligand-receptor assay, or the substrate of an enzyme).
Binding-pair assays rely upon the binding of one analyte by its specific binding partner to determine the concentration of the analyte within the test sample. Binding-pair assays may be differentiated and categorized as either competitive or non-competitive in nature. Non-competitive assays generally utilize the receptor component in a substantial excess over the concentration of the analyte to be determined in the assay.
One type of non-competitive assay is usually referred to as "sandwich assay." It employs the methodology whereby the analyte is detected via its binding to two binding partners. One partner may be labeled to facilitate a subsequent detection and the other is immobilized to a solid-phase to facilitate separation of the bound analytes from unbound reaction components (e.g., unbound labeled first receptor). An alternate non-competitive assay can be termed a "blocking assay." In this type of assay, sample is first mixed with a binding partner (usually labeled), and any analyte in the sample binds to the binding partner. The mixture is then allowed to react with analyte analog, which is usually bound to a solid phase. The more analyte is present in the sample, the more binding sites on the binding partner will be blocked, and the less sites there will be for the analyte analog to bind. Thus, in this form of assay, the more label is bound to the solid phase, the less analyte is present in the test sample.
In contrast, competitive binding-pair assays generally involve analyte from the test sample, a purified binding partner or binding partner analog that is labeled to facilitate detection, and a rate-limiting concentration of binding-partner species. The sample analyte and the labeled analyte/analyte analog moieties are subsequently allowed to compete for the limited number of binding sites provided by the binding partner species present in the assay mixture.
Competitive binding-pair assays can be further differentiated as being homogeneous or heterogeneous in nature. In homogeneous assays, all of the reactants participating in the competition reaction are mixed together and the concentration of analyte is determined by its effect on the extent of binding between its binding partner and labeled analyte. The signal observed is a direct function of this binding and can be related to the overall concentration of analytes present in the test sample. U.S. Pat. No. 3,817,837, which is incorporated herein by reference, discloses a homogeneous, competitive immunometric assay in which the labeled analyte analog is a ligand-enzyme conjugate and the binding partner is an antibody capable of binding either the analyte or analyte analog. In general, homogeneous assay systems require both an external instrumentality to determine the result and the prior calibration of the observed signal by separate tests performed with known concentration of the specific analyte in a process known as standardization. While homogeneous assays have dominated competitive immunometric assay system development, such systems are not capable of providing results for the determination of multiple analytes in a test sample in a single-test format not requiring external instrumentality.
Heterogeneous, competitive binding-pair assays require separation of the bound, labeled analyte or its binding partner from the free, labeled analyte or its binding partner and a subsequent measurement of either the concentration of the bound or free fraction. Methodologies for performing these assays are described in U.S. Pat. Nos. 3,654,090, 4,298,685, and 4,506,009, which are incorporated herein by reference. The quantitative or semi-quantitative measurement of analyte concentration utilizing this methodology cannot be performed without the use of additional tests to calibrate the assay results. Hence, only the presence or absence of the analyte can be determined without additional instrumentation or tests. Recently, however, methods have been developed for the internal calibration of binding-pair assays by providing a device which incorporates reference zones whereby the given response at the reference zone represents the assay response for a specific concentration of the analyte. The response generated by the unknown concentration of the analyte in the test sample is then compared with the response at the reference zone to determine the concentration of the analyte in the test sample in a qualitative or quantitative manner. U.S. Pat. No. 4,540,659, which is incorporated herein by reference, describes the system that incorporates several analyte concentration standards. They provide the ability to make semi-quantitative determinations of analyte concentrations in competitive binding-pair assays through a direct visual examination.
Sample collection and application means are known within the relevant field. For example, an applicator component, consisting of a separate wand-like component with a bibulous material attached to one end, is described in U.S. Pat. Nos. 5,169,789 and 4,770,853, which are incorporated herein by reference. The sample is collected for assay by simple absorption of the aqueous sample and subsequent placement of the collection component into the assay device. U.S. Pat. No. 4,624,929, incorporated herein by reference, discloses a sample collector comprised of a bibulous membrane confined in a housing. Collection of the sample is facilitated by contacting the sample collector with the desired aqueous sample. European Patent Application Nos. 88303744.2 and 90301697.0, incorporated herein by reference, disclose a wick-like sample collector, comprised of bibulous material, which is contiguous with the internal chromatography material.
While there are numerous assay devices and sample collection and application means that are currently in use within the relevant fields, the device disclosed herein serves to mitigate several of the difficulties which are frequently encountered in the utilization of these devices. For example, the requirement for large initial sample volumes, sometimes as much as several milliliters, often becomes problematic with many commercially available devices. This volume requirement is a function of the comparatively inefficient sample collection and/or application means these devices possess. Moreover, the requirement for such large initial sample volumes can potentially lead to difficulties when only small sample volumes or only a single test sample is available.
In contrast, due to the utilization of a novel sample collector and applicator, the present invention requires comparatively small initial sample volumes for analysis of analyte concentration. The features of the present invention also negate the need for secondary, external sample collector/applicator, which many, if not all, of the currently utilized devices require. Furthermore, the present invention greatly reduces the probability of sample contamination or cross-contamination, which is frequently encountered with the use of such secondary means. An additional unique feature of the sample collector/applicator component of the present device is the ability to collect and solubilize a dried sample, which putatively contains the analyte, without the use of secondary instrumentalities or methodologies.